Process of obtaining thevetin from the seeds of thevetia nerhfolia juss. or exile nut



Patented Nov. 26, 1935 UNITED STATES PATENT OFFICE K Kuei Chen and Amy Lin Chen, Indianapolis,

Ind., assignors to Eli Lilly and Company, Indianapolis, bid, a corporation of Indiana No Drawing. Application April is, 19.34; Serial No. 720,810

7 Claims.

It is the principal object of our invention to obtain in substantially pure form the active principle Thevetin, of the nuts or seeds of the- Thevetia neriifolia Juss, known variously as Exile oleander, Ahouai, Snake nut", "Joro- Joro", Be-still tree, and other names; and incidentally to obtain separately certain other substances, previously unknown, particularly Ahouain", Kokilphin", and a phytosterolin, all

10 co-present with thevetin in these nuts. For convenience we shall refer to the tree as exile oleander, and to the nuts as exile nuts.

- Thevetin, the active principle of these exile nuts,

is a heart stimulant of the general digitalis type.

10 The fundamental diflerence is that thevetin has a prompt action, and 'one of relatively shorter duration, while digitalis has a delayed action which starts only after a latent period and has a relatively longer duration.

The exile oleander is indigenous to South America and the West Indies; but is now cultivated in the East'Indies, India,- the Hawaiian Islands, and western Africa. It is not the same as the common'oleander of medicine-the nerium oleander. Its nuts are brown or black. The toxic property of these nuts has been known to European scientists since the latter part of the 16th obtained a pure thevetin, capable of producing,

consistent quantitative results either chemically or pharmacologically.

40 In obtaining our thevetin, we have proceededin general as follows:. The Exile-nut ker'nelspobtained by cracking and removing the shells, are crushed and/or ground, and macerated with ether for one or two days to defat them. The ether extract is separated, as by decantation. For increased defatting, the solid residue is ground more finely, and the ground material is then re-extracted with ether, desirably to exhaustion, as by continuous percolating in-a Soxhlet extractor.

The ether extract or extracts may be discarded, if thevetin alone is wanted, for it contains substantially no thevetin. However, it does contain a substance which is almost certainly a phytosterolin; and if that is desired, the ether ex- 5 tract, or combined ether extracts if several are made and they are combined, may be processed further; as will be explained hereinafter.

The solid residue or marc remaining after the ether extraction contains substantially all the de- 10 sired thevetin; and also some other things, although they are of less importance. This solid residue or marc is very irritating to the nose, and'causes The .thevetin in this thevetin-containing resi- 15 due or marc may be obtained as follows:

Desirably after such marc has been exhausted of .fats by the ether extraction above describedalthough complete exhaustion is not essential,

and indeed the entireether extraction may if de- 20 Thevetin The thevetin-containing liquid, after being separated from the sediment, is subjected to fraction'al precipitation with ether; which is added carefully, in small quantities, and the separate precipitates removed, as by filtration, until the) addition of ether causes no further precipitation. The precipitates,. preferably separately, are washed in ether, dried in vacuum, powdered in a mortar, and dissolved in the smallest possible amount of warm isopropyl alcohol. when 45 these alcoholic solutions are allowed to stand at room temperature, thevetin appears .as a fine white powder, which can be separated by filtration under suction. While it is possible to combine the fractional precipitates, and to treat them 50 together, we prefer to treat them separately; as the last few fractions of the precipitate yield thevetln more quickly than do the first or second fractions. The crude thevetin which is obtained by this treatment of the fractional precipitates is combined; and the combined crude thevetln is purified by repeated recrystallization (twice usually being sufiicient) from 75% to 90% isopropyl alcohol.

Thevetin crystallizes in clusters from 90% isopropyl alcohol, and in fine needles from 75% to 80% isopropyl alcohol. It is soluble in pyridine, methyl alcohol, ethyl alcohol, and n-propyl and isopropyl alcohols; soluble in both cold and warm water, but less so than in alcohol; and practically insoluble in ether, acetone, chloroform, and benzene. It melts at about 193 C. (corrected). A 2% solution in methyl alcohol has been found to give a polarimetric reading of -2.5 in a 2- decimetertube, with sodium light; from which the specific rotation of thevetin is determined as On being treated with concentrated sulphuric acid, thevetln produces a yellow color, which turns to purple; with concentrated hydrochloric acid, a light yellow color, which changes to bluish green; and with concentrated nitric acid, a light yellow color, which persists. It gives a. positive test with sodium nitroprusside or with Tollens reagent. It gives a negative reaction in the Keller-Killiani test; which seems to indicate the absence from the thevetin molecule 'of any desoxy sugar, such .as digitoxose or cymarose. Its

Air-dry substance 4.868 mgm.; 0.276 mgm. loss of weight 5.073 mgm.: 0.269 mgm. loss of weight 5.061 mgm.: 0.276 mgm. loss of weight 4.848 mgm.: 0.269 mgm. loss of weight Calculated" H 0 5.647 Found "Hi0 5.67, 5.50, 5.45, 5.55%. Anhydrous substance 4.804 mgm.: 10.155 mgm. C02, 3.270 mgm. H2O

9.410 mgm. C02, 3.010 mgm. H2O 9.440 mgm. CO2, 3.000 mgm. H2O

camphor: 4.7 A camphor: 5.9 A camphor: 4.5 A

4.542 mgm.: 4.450 mgm.:

0.300 mgm. in 4.260 mgm. 0.559 mgm. in 6.270 mgm. 0.312 mgm. in 4.630 mgm.

Molecular weight calculated 602 Molecular weight found 600, 605, 599

The empirical formula is not asserted positively, for its correctness will depend upon the outcome .of a study of the sugar portion and of the genin' (non-sugar) molecule.

Hydrolysis of thevetin according to themethod portion of the cardiac-glucoside of Windaus and Hermanns (Ber. Deut. Chem. Gesellsch., 48, 979, year 1915) results in the formation of a resinous material. The supernatant fluid, upon neutralization with silver carbonate, reacts with a-naphthol and concentrated sul phuric acid, to form a reddish purple ring. With a solution of orcinol and hydrochloric acid (Bials reagent) this supernatant fluid yields a brown color, and forms a precipitate; which indicates that the sugar portion of the glucoside is not likely a simple pentose.

The pharmacological eflects of thevetln upon the amphibian and mammalian heart are in general like those of digitalis; but more prompt in starting and shorter in duration.

In frogs, thevetln in small doses (0002-0004 mgm. per gm. injected into the lymph sac has little efiect upon motor activity, but sometimes causes a slight restlessness. Somewhat larger amounts, similarly administered, usually cause systolic standstill of the ventricle in an hour. Doses of 0.05 to 0.1 mgm. per gm. result in a stupor.

By perfusion into the inferior vena cave of the frog, thevetln in a concentration of 1:20,000 to 1210,000 produces an initial augmentation of the ,ventricular contraction, butthis augmentation lasts only for one or two minutes and is followed by diminution of systole and diastole, especially diastole. It causes a diminution of the heart rate. Occasionally partial or complete A-V block and extra systoles appear. In twenty or thirty minutes after injection, in the concentration noted,

the ventricle stops at systole, while the auricle may continue beating for some time; and normal Ringer's solution is not able to restore the original cardiac activity.

Since thevetin is soluble in water, and is easily absorbed into the circulation, it can be easily standardized by the U. S. P. frog method. (The Pharmacopoeia of the United States, J. B. Lippincott Co., Philadelphia, 10th revision, 393, year 1926.) The minimal systolic dose for different lots is from 0.004 to 0.005 mgm. per gm.

In etherized cats, a dose of 1 mgm. per kgm. injected rapidlyby vein raises the arterial blood pressure, with a gradual return to the initial level. During the rise in blood pressure slowing of the heart rate occurs, followed by arrhythmia, as may be shown either by kymographic records or by auscultation. We have established a cat unit", which is the least amount per kilogram that on intravenous injection stops the heart-beat of an etherized cat. It is 0.85 mg. per kg. This cat unit is a convenient unit for use by clinicians. because they are familiar with it in connection with their administration of digitalis.

Electrocardiograms (taken from Lead II in cats) show that thevetln produces typical effects of digitalis-type intoxication during the injection of a 1:20,000 solution intravenously at the rate of 1 cc. per minute. Essential changes, in substantially the general order (which is not always quite the same) of their occurrence, are bradycardia, inversion of the T wave, P-R prolongation, ventricular rhythm, A-V dissociation, secondary tachycardia, and finally, ventricular'fibrillation;

, charring.

been used by Hanzlik for digitalis: and t y t for cats follows that which has been developed by us for digitalis-like substances.

When thevetin is incubated with sheeps redblood-cell corpuscles at 37 C. for an hour, hemolysis does not occur in thevetin concentrations ranging from 1:8000 to 1:500.

Pure thevetin is found,-not to cause necrosis at the site of injection; but, like other digitaloid glucosides, may produce slight irritation, chiefly manifested as a slight pain. I

By tabulating data available, it has been found that the average P-R prolongation occurred at 17%, maximal slowing of the heart at 30%, and ectopic rhythm at 31% of thefatal dose of thev- ,etin; as against P-R prolongation at 48%, maximal slowing at 56%, and ectopic rhythm at 61% of the fatal dose of digitoxin, and as against P-R. prolongation at 37%, maximal slowing at 58%, and ectopic rhythm at 54% of the fatal dose of ouabain. Thevetin thus has a more prompt action than has either digitoxin or ouabain, and

requires a smaller percentage of the fatal dose to produce digitalis-like eiiects.'

Thevetin has also been used in human casesparticularly in the treatment of auricular fibrillation and flutter, myocardial insufllciency, heart block, and hyperthyroidism with cardiac decompensat'ion. It acts generally like digitalis, but its efiect both appears and disappears more rapidly thandoes that of digitalis. Thevetin is eflective either whentaken by mouth or when injected intravenously. A combination of thevetin and digitalis ofiers the advantages of both thevetin and digitalis. As determined by tests on animals,

particularly on frogs, cats, and dogs, the'vetin is Phytosterolin The ether extract, or combined ether extracts if several are made, from the first extraction of the crushd exile-nut kernels is subjected to evaporation to remove the ether; which leaves behind a light yellow fatty oil. Such an oil has been obtained and studied to some extent by previous investigators.

We have found that when this fatty oil is allowed to stand at room temperature, a solid appears at the bottom and sides of the container--a solid which is usually at least partially in crystalline form. This is separated from the supernatant liquid, as by filtration, and washed with ether; which leaves awhite powder. powder may be purified by repeated recrystallization from a mixture of pyridine and alcohol; which procedure yields large opaque spheroidal crystals which melt at 291-292.5 C. (corrected) with The substance thus obtained gives a positive reaction with either the Liebermann-Burchard test or the Salkowski test; and upon hydrolyzing in a mixture of hydrochloric acid and alcohol in a sealed tube on a water bath, filtering, and neutraliaing the filtrate, such neutralized filtrate readily reduces Fehlings solution. It is almost certainly a phytosterolin. Elementary analyses v 3 m CnHuOLcHuOs (calculated C 72.20%. H 10.20%; found, on duplicate determinations, C 72.15% and 4.691 mg.:l2.405 mg. C02, 4.300 mg. mo

4.311 mg.:1'l'.400 mg. C01, 4.010 mg.

'Ahouain I The sediment which formed when the solution 10 in absolute ethyl alcohol was allowed to stand a over night contains chiefly ahouain and kokiiphin, as has already been stated. This sediment is treated with warm absolu ethyl alcohol. Although the sediment settled 15 from a solution made with warm absolute ethyl alcohol, the sediment does notat this later stage go wholly into solution, but leaves an undissol'ved residue. This residue and the supernatant liquid are suitably separated, as by decantation.

The residue contains chiefly ahouain; and the supernatant liquid contains chiefly kokilphin.

The ahouain-containing residue'is dissolved in methyl alcohol, and is subjected to fractional precipitation with chloroform. The .last fractional g5 precipitates obtained are chiefly ahouain; and these are crystallized from absolute methyl aloe-'- hol, in a desiccator saturated with ether vapor. We are not able to determine the molecular weight of ahouain by the East camphor method, 30

(Ber. Deut. Chem. Gesellsch, 55, 1051 year 1922,) nor by the boiling-point method in methyl alcohol. By determinations by the Barger method (Jour. Chem. Soc., 85, 286, year 1904) made for us by Dr. J. B. Whitman, yielded a set of relatively,

consistent values, with azobenzene as a standard and methyl-alcohol as the' solvent. 0n the basis of these determinations, and the results of combustion analyses, we believe that ahouain has the empirical formula 'CmHmOio (calculated C 40 a H 6.43% and 8.44%, mol. wt. 31'], 263,292, and

I 333). The actual analysis on which this is based is as follows:

4.664 mg.:6.845 mg. CO2, 2,680 mg. 4.188 mg.:6.150 mg. 0 4, 2.410 mg. mo.

Ahoualn has no action on the frog's heart in dosage of 0.5 mgm. per gm. administered Silba cutaneously. Intravenous injection of it in-rabbits, in doses of-2 mgm. per'kgm increases the blood sugar from 18 to 24 mgm. per 100 cc. of

blood.

1 lfolcilphin' The kokilphin-containing supernatant liquid which was separated from the ahouain-containing residue is concentrated. by vacuum distillation to a small volume. and benzene is gradually added 60 to the concentrate until no further precipitation occurs. On standing for some .time, crystals of kokilphin are deposited at the-bottom. .These are separated, as by decantation, and purified by recrystallization from absolute or ethyl alcoc5 I hol.

Kokilphin crystallizes from absolute ethyl alcohol in angular prisms which melt sharply at 188.5--189 C. (corrected). It is soluble in water and in methyl alcohol, less soluble in ethyl alco- 70'- hol, and almost insoluble in ethenbenzene. and. acetone. It does not show color displays with concentrated minerals acids, such as sulphuric, nitric, or hydrochloric acids.- When kokilphinis boiled with renim s solution or with Benedict's solution no apparent reaction takes place; but if a solution of kokilphin is previously hydrolyzed with an acid, and then treated'with the sugar reagents, reduction occurs.

jec ting such solutionto fractional precipitation with ether to produce a precipitate of thevetin;

3. The process of obtaining thevetin from the kernels of exile nuts, comprising extracting them Molecular weight determinations according to with methyl alcohol and evaporating such alco- 5 the Rest method and elementary analyses indiholic extract to dryness, redissolving the residue cate that kokilphin has the empirical formula after evaporation in absolute ethyl alcohol, al- C33H61O3o (calculated,C42.25%,H6.56% m0l1,wt. lowing the alcoholic solution to stand until a 937; found, on duplicate determinations, C 42.04 sediment forms, and separating that sediment and 42.21%, H 6.51 and 6.53%, mol. wt. 935 and from the supernatant. liquid, and subjecting such 953) The actual analysis on which this is based supernatant liquid to fractional precipitation is as follows: with ether to produce a precipitate of thevetin.

4.490 mg.:6.920 mg. CO2, 2.610 mg. H 4. The process of obtaining thevetin from the 4.521 mg.:6.995 mg. CO2, 2.640 mg. H20 kernels of exile nuts, comprising extracting them 0-208 mg. i 3-070 P with methyl alcohol and evaporating such alco- 5 0-245 11! 3-120 mg. camp 0 A- holic extract to dryness, redissolving the residue In fmgs, kokflphin causes no change in cardiac after evaporation in absolute ethyl alcohol, and activity on subcutaneous injection of 4.0 mgm. subjecting such m to fractional precipita" per In doses of 2 mgm per kgm" admims tion with ether to produce a precipitate of tered intravenously in rabbits, it produces a slight thevetm- 20 hyperglycemia; the increase in blood sugar in two 5. The I'M'DCGSS- Of obtaining thevetin from the experiments not exceeding 11 to 14 mgm. per 100 k r ls f exile nuts, p is d attins them. cc. of blood. forming an alcoholic solution of the defatted ker- To facilitate understanding of the steps of the nels, al S Solutifln Stand until a sedl- 25 specific process which is described by way of ment appears, separating that sediment from example, the following flow sheet will be of the supernatant liquid, and precipitating thevetin service: from the supernatant liquid with ether.

Flow sheet Exile-nut-kernels etllier filt ata residue (marc) evap orate methyl alcohol fatty oil evaporate to dryness stand warm absolute ethyl alcohol crystals of a phytosteroliu stalnd 40 sedl!:nent supernat ant liquid warm absoluteethyl alcohol Iractional precipitate with other remide isoprop lalcohol sediment or white powder (thevetin) concentrated by vacuum distillation methyl alcohol mum t I m i l a ho! 1'8 sedimen precipitate kokitphin with benzene precipitate ahoualn with chloroiorm ms to wimpy m recrystallize from or absolute ethyl alcohol We claim as our invention:

1. The process of obtaining thevetin from the kernels of exile nuts, comprising defatting them with ether, extracting the residue or marc with methyl alcohol and evaporating such alcoholic extract to dryness, redissolving the residue after evaporation in absolute ethyl alcohol, allowing the alcoholic solution to stand until a sediment forms, and separating that sediment from the supernatant liquid, and subjecting such supernatant liquid to fractional precipitation with ether to produce a precipitate of thevetin.

2. The process of obtaining thevetin from the kernels of exile nuts, comprising defatting them with'ether, extracting the residue or marc with methyl alcohol and evaporating such alcoholic extractto dryness, redissolving the residue after evaporation in absolute ethyl alcohol, and sub recrystallizel from absolute methyl alcohol 6. The process of obtaining thevetin from the kernels of exile nuts, comprising forming an alcoholic solution of the kernels, allowing such solution to stand until a sediment appears, separating that sediment from the supernatant liquid. and precipitating thevetin from the supernatant liquid with ether. l

7. The process of obtaining thevetin from the kernels of exile nuts, comprising forming an alcoholic solution of the kernels, allowing such solu tion to stand until a sediment appears, separating that sediment from the supernatant liquid, precipitating thevetin from the supernatant liquid with ether, and purifying such precipitate by recrystallization from isopropyl alcohol.

KO KUEI CHEN.

AMY LING CHEN. 

